Of the five IE proteins of HSV-1, the role of ICP22 during lytic infection is least well understood. Although ICP22 was initially characterized as being dispensable for virus replication, it was later shown that ICP22 expression is absolutely required for productive infection in many cultured cell types and in vivo. The region of the HSV-1 genome encoding ICP22, US1, also encodes a second protein, ppUS1.5, that initiates within the US10RF and is colinear with the ICP22 protein. Because all initial studies with ICP22 mutants were accomplished with ICP22/ppUS1.5 double mutants, it is unclear at this time whether one or both of these proteins is required for the phenotypes associated with US1-/US1.5- viruses. The three ICP22-/ppUS1.5- double mutant viruses studied to date exhibit impaired a) replication in some cell types (restrictive) but not in others (permissive), b) viral gene expression, and c) establishment and reactivation of latency. To determine the functional requirements for ICP22 and ppUS1.5 in the HSV-1 life-cycle, we will generate HSV-1 mutant viruses that do not express ICP22 or ppUS1.5, and inducible retrovirus expression vectors that express only these proteins. These reagents will be used to test the roles of ICP22 and ppUS1.5 in establishing the published phenotypes of ICP22-/ppUS1.5- mutants--including the ability to inhibit the transactivating activity of another IE protein, ICP0. The studies outlined in this proposal are designed to elucidate the roles of ICP22 and ppUS1.5 in productive infection and ultimately, in HSV-1-induced disease.